HIF-1α-糖酵解通路在丙泊酚减轻急性睡眠剥夺老龄大鼠脑损伤中的作用

Role of HIF-1α-glycolytic pathway in propofol-induced attenuation of brain injury in acute sleep-deprived aged rats

目的:评价缺氧诱导因子-1α(HIF-1α)-糖酵解通路在丙泊酚减轻急性睡眠剥夺老龄大鼠脑损伤中的作用。方法:SPF级健康雄性SD大鼠48只,20~22月龄,体质量500~600 g,采用随机数字表法分为4组( n=12):对照组(Con组)、睡眠剥夺组(SD组)、睡眠剥夺+丙泊酚组(SP组)和睡眠剥夺+丙泊酚+二甲基乙二酰氨基乙酸(DMOG)组(SPD组)。采用睡眠剥夺仪建立睡眠剥夺模型。于睡眠剥夺前20和3 h时,SP组腹腔注射1%丙泊酚0.04 mg/g,SPD组腹腔注射1%丙泊酚0.04 mg/g和HIF-1α特异性激动剂DMOG 0.05 mg/g。采用条件恐惧实验、新旧物体识别实验评估大鼠记忆能力。行为学实验结束后,取血液和脑组织标本,采用伊文思蓝(EB)染色法检测血脑屏障通透性,HE染色后观察海马组织病理学变化,TUNEL染色法确定海马神经元凋亡率,ELISA法检测血清神经丝轻链蛋白(NfL)、NSE浓度以及海马组织ROS、IL-6、诱导型一氧化氮合酶(iNOS)、乳酸和丙酮酸含量,计算乳酸/丙酮酸比值;采用Western blot法检测海马HIF-1α、丙酮酸激酶M2 (PKM2)、己糖激酶2(HK2)、离子钙结合适配器分子1(IBA1)以及裂解的半胱氨酸天冬氨酸蛋白酶3(cleaved caspase-3)的表达。 结果:与Con组相比,SD组和SPD组僵直时间百分比和识别指数降低,血清NSE和NfL浓度、脑组织EB含量、海马组织ROS、IL-6、iNOS含量和乳酸/丙酮酸比值升高,HIF-1α、PKM2、HK2、IBA1、cleaved caspase-3表达上调,神经元凋亡率升高( P<0.05),海马区神经细胞发生病理学损伤。与SD组相比,SP组僵直时间百分比和识别指数升高,血清NSE和NfL浓度、脑组织EB含量、海马组织ROS、IL-6、iNOS含量和乳酸/丙酮酸比值降低,HIF-1α、PKM2、HK2、IBA1、cleaved caspase-3表达下调,神经元凋亡率降低( P<0.05),海马神经细胞病理学损伤减轻。与SP组相比,SPD组僵直时间百分比和识别指数降低,血清NSE和NfL浓度、脑组织EB含量、海马组织ROS、IL-6及iNOS含量、乳酸/丙酮酸比值升高,HIF-1α、PKM2、HK2、IBA1、cleaved caspase-3表达上调,神经元凋亡率升高( P<0.05),海马区神经细胞病理学损伤加重。 结论:丙泊酚可减轻急性睡眠剥夺诱导的老龄大鼠脑损伤,其机制可能与抑制HIF-1α-糖酵解通路,减轻神经炎症反应有关。

Objective To evaluate the role of hypoxia-inducible factor 1α(HIF-1α)-glycolytic pathway in propofol-induced attenuation of brain injury in acute sleep-deprived aged rats.Methods Forty-eight male SPF-grade healthy Sprague-Dawley rats,aged 20-22 weeks,weighing 500-600 g,were divided into 4 groups(n=12 each)using a random number table method:control group(Con group),sleep deprivation group(SD group),sleep deprivation+propofol group(SP group),and sleep deprivation+propofol+DMOG group(SPD group).The sleep deprivation model was developed using a sleep deprivation apparatus in rats.At 20 and 3 h before sleep deprivation,1%propofol at a dose of 0.04 mg/g was intraperitoneally injected in SP group,and 1%propofol at a dose of 0.04 mg/g and HIF-1αactivator DMOG at a dose of 0.05 mg/g were intraperitoneally injected in SPD group.Cognitive function was assessed by fear conditioning test and novel object recognition test.After the behavioral testing,the blood and brain tissue samples were collected for determination of blood-brain barrier permeability(by Evans blue staining),apoptosis rate(by TUNEL staining),serum levels of neurofilament light chain protein(NfL)and neuron-specific enolase(NSE),contents of reactive oxygen species(ROS),interleukin-6(IL-6),inducible nitric oxide synthase(iNOS),lactate and pyruvate(by enzyme-linked immunosorbent assay)and expression of HIF-1α,pyruvate kinase M2(PKM2),hexokinase 2(HK2),ionized calcium-binding adaptor molecule 1(IBA1),and cleaved caspase-3(by Western blot)and for microscopic examination of pathological changes of the hippocampal tissues.The lactate/pyruvate ratio was calculated.Results Compared to Con group,the percentage of freezing time and recognition index were significantly decreased,serum concentrations of NSE and NfL,EB content in brain tissues,contents of ROS,IL-6 and iNOS in hippocampal tissues,and lactate/pyruvate ratio were increased,the expression of HIF-1α,PKM2,HK2,IBA1 and cleaved caspase-3 was up-regulated,the apoptosis rate was increased(P<0.05),and the pathological damage to nerve cells in the hippocampus was found in SD group and SPD group.Compared to SD group,the percentage of freezing time and recognition index were significantly increased,serum concentrations of NSE and NfL,EB content in brain tissues,contents of ROS,IL-6 and iNOS in hippocampal tissues,and lactate/pyruvate ratio were decreased,the expression of HIF-1α,PKM2,HK2,IBA1 and cleaved caspase-3 was down-regulated,the apoptosis rate was decreased(P<0.05),and the pathological damage to nerve cells in the hippocampus was significantly attenuated in SP group.Compared to SP group,the percentage of freezing time and recognition index were significantly decreased,the serum concentrations of NSE and NfL,EB content in brain tissues,contents of ROS,IL-6 and iNOS in hippocampal tissues,and lactate/pyruvate ratio were increased,the expression of HIF-1α,PKM2,HK2,IBA1 and cleaved caspase-3 was up-regulated,the apoptosis rate was increased(P<0.05),and the pathological damage to nerve cells in the hippocampus was aggravated in SPD group.Conclusions Propofol can attenuate acute sleep deprivation-induced brain injury in aged rats,and the mechanism may be related to inhibition of the HIF-1α-glycolytic pathway and reduction of neuroinflammatory responses.